The anti DEC205 antibody (?DEC205) recognizes a surface protein on dendritic cells (DCs) that can be used to target antigens to DCs: attachment of an antigenic moiety to ?DEC205, either through chemical conjugation or as a genetic fusion, allows its delivery to the DC. This generates a potent adaptive immune response that includes CD4 and CD8 T cells specific for the antigen attached to?DEC205. This approach -as well as similar modifications of other antibodies directed against yet other surface proteins on dendritic cells, such as Class II MHC products- holds promise as a possible vaccine strategy. The lack of precision of most chemical conjugation methods, and the labor-intensive genetic fusion approaches required to attach the payload of interest to ?DEC205 and purify the resulting adduct suggest an alternative to eliminate these shortcomings. A highly efficient and straightforward chemo-enzymatic alternative, using sortase from Staphylococcus aureus, is proposed to allow a detailed analysis of the mechanisms that underlie the desirable immunogenic properties of ?DEC205 adducts. A comparison will be made with Vhh7, a single domain anti- mouse Class II MHC antibody derived from an alpaca. Vhh7 was engineered in similar fashion to serve as a sortase substrate, validated through generation of fluorescent and biotinylated derivatives as the means of demonstrating its specificity and tight binding to Class I MHC products The introduction of a LPXTG motif at the C-terminus of the heavy chain of ?DEC205 or onto Vhh7 allows site-specific attachment of a T cell epitope, a fluorescent or biotinylated payload of choice and even protein-sized substituents in a stoichiometric manner. This procedure, referred to as sortagging, should permit the specific delivery of any T cell epitope or traceable payload (including protein-sized attachments) to DEC205+ and Class II MHC+ antigen presenting cells in vivo, without the need for numerous independent genetic ?DEC205 or Vhh7 fusion constructs. The proposed method has the added advantage that non-natural substituents, such as easily cleavable linkers or adjuvants, can be installed to improve potency of such adducts, and that even C-terminus to C-terminus fusions are possible -using click chemistry- to compare efficacy of presentation of C-N versus C-C fusions of ?DEC205 or Vhh7 with antigen. These goals will be addressed in two specific aims, to be applied to CD8 T cells, specific for the MHV68 ORF8 antigen, obtained from cloned mice created by somatic cell nuclear transfer: 1. Establish the mechanism that underlies ?DEC205- and Vhh7 dependent routes of antigen cross- presentation. 2. Explore the ability of ?DEC205 and VHH7 adducts to activate effector CD8 T cells (or elicit them de novo) and establish protection against MHV68 virus. If successful these strategies would open the door to creating desirable T cell responses without the use of infectious agents and generally improve options for immune manipulations to achieve therapeutic effect. ! PUBLIC HEALTH RELEVANCE: The DEC205 antibody, and an alpaca derived single domain antibody fragment, VHH7, will be engineered to receive, in a simple enzymatic reaction, an antigen of interest modified with chemical tags that will allow a detailed analysis of the attached antigenic cargo. The ability of such adducts to elicit a desirable CD8 T cell response against an infectious agent, MHV68, will be examined as a step towards developing this platform in a generalizable and practical vaccine strategy.